Use of an active ingredient for the manufacture of a product for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization with Corynebacterium glucosidilyticum or a close relative thereof

Abstract

The present application provides a kind of active ingredient in the preparation for preventing and / or treating the use of product caused by Corynebacterium glucosidium or its close relative colonization, calcium oxalate kidney stone, the active ingredient is selected from one or several of the following substances: hippuric acid, pharmaceutically acceptable salt of hippuric acid, hippurate prodrug, benzoic acid or its pharmaceutically acceptable salt, and the substance capable of promoting the synthesis of hippuric acid in vivo.Hippuric acid supplementation or the intervention of cranberry diet can significantly increase the level of urine hippuric acid, effectively inhibit the formation and aggregation of calcium oxalate crystals induced by Corynebacterium glucosidium or its close relative.The present application also provides a pharmaceutical composition and kit comprising the above active ingredient and a qPCR method for detecting Corynebacterium glucosidium or its close relative for non-diagnostic purposes;The present application provides a new pathogen target and treatment strategy for the prevention and treatment of calcium oxalate kidney stone, which has significant clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the use of an active ingredient in the preparation of products for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of Corynebacterium glutamicum or its closely related species. Background Technology

[0002] Kidney stones are one of the most common diseases in urology, and their exact pathogenesis is not yet fully understood. In recent decades, the global prevalence of kidney stones has been steadily increasing. my country is one of the three highest-incidence areas for kidney stones in the world. Epidemiological surveys at different times show that the prevalence of urinary tract stones was 6.6% before 2010, 7.3% from 2011 to 2015, and 10.5% from 2016 to 2020, respectively. Currently, kidney stones are considered a systemic disease, associated with cardiovascular disease, hypertension, obesity, metabolic syndrome, chronic kidney disease, and osteoporosis. They not only seriously affect patients' quality of life but also impose a heavy socioeconomic burden. Calcium oxalate stones are the main type of kidney stone, accounting for approximately 70%-80%. Their formation involves multiple processes, including supersaturation of calcium and oxalate ions in urine, nucleation, growth, aggregation, and adhesion of calcium oxalate crystals. Hypercalciuria and hyperoxaluria caused by endogenous factors such as metabolism and genetics are common risk factors for the development of calcium oxalate stones. Statistics show that the 5-year recurrence rate of kidney stones is 50%, while the 10-year recurrence rate is as high as 80%-90%. Although minimally invasive surgical techniques such as laser lithotripsy can effectively remove stones, the formation mechanism of calcium oxalate stones is not yet clear, and there are still significant scientific challenges in early prevention and postoperative recurrence control.

[0003] Urinary tract microorganisms are closely related to calcium oxalate stones. Urinary microorganisms may directly participate in regulating urine composition, thus directly affecting the occurrence and development of calcium oxalate stones. Currently, urinary tract infection caused by Escherichia coli is of the highest concern. However, most patients with calcium oxalate stones do not have typical urinary tract infection symptoms, and antibiotic use is also considered a risk factor for stone formation. This suggests that microorganisms may influence stone formation through non-classical infection routes.

[0004] A series of factors regulate and inhibit crystal formation in urine. Currently, it is believed that the main stone-inhibiting factors include citrate, succinate, and Mg²⁺. + Osteopontin, lecithin, and glycosaminoglycans, etc.; while stone-forming factors are mainly composed of Ca²⁺. + Primarily oxalic acid. A growing body of research indicates that the levels of these factors are significantly influenced by host-microbe co-metabolic networks. For example, oxalic acid-degrading bacteria in the gut (such as...) Oxalobacter formigenesIt can metabolize oxalate in the intestines, reducing its reabsorption and lowering oxalate levels in urine, thus inhibiting the formation of calcium oxalate stones. However, its clinical efficacy as a "probiotic" for treating calcium oxalate stones remains controversial. Notably, studies have shown a stronger correlation between urinary microbiome and kidney stone formation than with gut microbiota. Unlike the indirect effects of gut microbiota, urinary microbiota may directly participate in changes in urine composition, thus more directly influencing the occurrence and development of calcium oxalate stones. However, due to the low biomass of urinary microorganisms and the limited number of species that can be identified by traditional culture and conventional sequencing techniques, current research is mostly limited to comparing the differences in urinary microbiota structure between stone patients and healthy individuals. The relevant mechanisms of action remain unclear, and overall research in this field is still insufficient. Summary of the Invention

[0005] In view of this, the present invention provides the use of an active ingredient in the preparation of a product for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of *Corynebacterium glutamicum* or its closely related species. This invention discloses for the first time the use of *Corynebacterium glutamicum* (… Corynebacterium glucuronolyticum The inventors investigated the causal relationship between *Corynebacterium glutamicum* or its close relatives and the development of calcium oxalate kidney stones. Through analysis of clinical samples, the inventors found that the detection rate of *Corynebacterium glutamicum* or its close relatives in the urine of patients with calcium oxalate stones was significantly higher than in healthy individuals. Furthermore, this bacterium possesses hippuric acid hydrolase activity, enabling it to degrade hippuric acid in urine, which has the function of inhibiting the formation of calcium oxalate crystals.

[0006] The *Corynebacterium glucoside* or its closely related species as described in this invention refer to a group of rod-shaped bacilli that possess hippuric acid hydrolase activity and can promote the formation of calcium oxalate crystals; including *Corynebacterium glucoside*. (Corynebacterium) glucuronolyticum) , as well as closely related species that are phylogenetically related.

[0007] The objective of this invention is achieved through the following technical solution: First aspect: The present invention provides the use of an active ingredient in the preparation of a product for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of Corynebacterium glutamicum or its closely related species. The active ingredient is selected from one or more of the following substances: hippuric acid, pharmaceutically acceptable salts of hippuric acid, hippuric acid ester prodrugs, benzoic acid, pharmaceutically acceptable salts of benzoic acid, and substances capable of promoting the synthesis of hippuric acid in vivo.

[0008] The plant extract that promotes the synthesis of hippuric acid in the body is a plant extract rich in hippuric acid, benzoic acid, benzoic acid glycosides, or polyphenolic compounds.

[0009] The plant extract is derived from one or more of the following: cranberry, blueberry, European blueberry, raspberry, cloudberry, redberry, plum, plum, and jujube.

[0010] Pharmaceutically acceptable salts of benzoic acid include sodium benzoate or potassium benzoate.

[0011] The products are oral tablets, sustained-release granules, liquid preparations for injection or perfusion, and functional foods.

[0012] The above-mentioned active ingredients effectively prevent or treat calcium oxalate kidney stones caused by colonization of Corynebacterium glutamicum or its closely related species by increasing the hippuric acid content in urine and restoring the inhibitory effect of hippuric acid on the formation of calcium oxalate crystals.

[0013] A rat infection model confirmed that direct supplementation with hippuric acid or a cranberry diet that promotes hippuric acid synthesis can significantly reduce the number of calcium oxalate crystals in the urine of rats infected with Corynebacterium glutamicum or its close relatives and eliminate crystal aggregation. At the same time, it can significantly increase the level of hippuric acid in urine and blood, verifying the effectiveness of the above-mentioned active ingredients.

[0014] The *Corynebacterium glucoside* or its closely related species as described in this invention refer to a group of rod-shaped bacilli that possess hippuric acid hydrolase activity and can promote the formation of calcium oxalate crystals; including *Corynebacterium glucoside*. (Corynebacterium) glucuronolyticum) And closely related species that are phylogenetically related. This includes, but is not limited to: *Corynebacterium glucoside* N11, N8, N10, N28, the type strain DSM 44120, and *Corynebacterium paraglucoside*. (Corynebacterium) paraglucuronolyticum) N9, etc. It should be noted that the above-mentioned strains are only representative isolates of the present invention. Those skilled in the art can isolate other functionally equivalent strains with equivalent hippuric acid hydrolase activity and pathogenicity from the urine of patients with calcium oxalate stones using conventional microbiological methods. These strains also fall within the scope of "Corynebacterium glutamicum or its closely related species" as described in the present invention.

[0015] Among them, Corynebacterium glucoside N11 ( Corynebacterium glucuronolyticum This strain (N11) was deposited at the China Center for Type Culture Collection on April 30, 2026, with accession number CCTCC NO: M2026881.

[0016] Second aspect: a pharmaceutical composition comprising one or more of the following substances: hippuric acid, a pharmaceutically acceptable salt of hippuric acid, a hippuric acid ester prodrug, benzoic acid, a pharmaceutically acceptable salt of benzoic acid, and a substance capable of promoting the synthesis of hippuric acid in vivo; said composition is used for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of Corynebacterium glutamicum or its closely related species.

[0017] The plant extract that promotes the synthesis of hippuric acid in the body is a plant extract rich in hippuric acid, benzoic acid, benzoic acid glycosides, or polyphenolic compounds. The plant extract is derived from one or more of cranberries, blueberries, bilberries, raspberries, cloudberries, cranberries, plums, green plums, and jujubes. Pharmaceutically acceptable salts of benzoic acid include sodium benzoate or potassium benzoate.

[0018] The pharmaceutical composition further includes pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients are selected from one or more of fillers, binders, disintegrants, lubricants, coating materials, solvents, solubilizers, preservatives, and flavoring agents.

[0019] Third aspect: A kit for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of *Corynebacterium glutamicum* or its close relatives, said kit comprising: a) hippuric acid, its pharmaceutically acceptable salt, or its ester prodrug; or b) Substances that can promote the synthesis of hippuric acid in the body.

[0020] Fourth aspect: A method for screening candidate drugs for the prevention and / or treatment of calcium oxalate kidney stones, characterized by comprising the following steps: (1) Contact the candidate material with Corynebacterium glucoside or its closely related species, and / or its expressed hippuric acid hydrolase; (2) Detect the activity of the hippuric acid hydrolase; If the activity of the hippuric acid hydrolase is inhibited, it indicates that the candidate substance can be used for the prevention and / or treatment of calcium oxalate kidney stones.

[0021] Preferably, the effectiveness of the candidate substance is further confirmed if an increase in hippuric acid levels and / or a return to normal levels of the optional metabolites are detected simultaneously.

[0022] Fifth aspect: A qPCR method for detecting *Corynebacterium Glucono-deltae* or its close relatives for non-diagnostic purposes, comprising the following steps: (1) Extract DNA from the sample to be tested; (2) Using the extracted DNA as a template, qPCR amplification was performed using the forward primer shown in SEQ ID NO.33, the reverse primer shown in SEQ ID NO.5, and the TaqMan fluorescent probe shown in SEQ ID NO.32; (3) Determine whether there is *Corynebacterium glutamicum* or its closely related species in the sample to be tested based on the amplification curve.

[0023] The sample to be tested is a urine sample; the specific operation of DNA extraction in step (1) is as follows: collect the urine sample, centrifuge to collect the precipitate, extract DNA using a urine DNA extraction kit, and wash with preheated nuclease-free water.

[0024] The qPCR amplification reaction system is 20 μL, containing: 10 μL of 2×AceQ Universal U Probe MasterMix V2, 0.4 μL of 10 μM forward primer, 0.4 μL of 10 μM reverse primer, 0.2 μL of 10 μM TaqMan probe, 2 μL of DNA template, and 7 μL of nuclease-free water.

[0025] The qPCR amplification program includes: 37°C contamination digestion for 2 min; 95°C pre-denaturation for 5 min; and 40 cycles of 95°C denaturation for 10 sec, 60°C annealing and extension for 30 sec.

[0026] Sixth aspect: A combination of oligonucleotides for detecting *Corynebacterium Glucono-deltae* or its close relatives, comprising: The forward primer has the nucleotide sequence shown in SEQ ID NO.33; The reverse primer, whose nucleotide sequence is shown in SEQ ID NO.5; and The TaqMan fluorescent probe has the nucleotide sequence shown in SEQ ID NO.32, and the 5' end of the probe is labeled with a FAM fluorescent reporter group and the 3' end is labeled with an MGB quencher group.

[0027] Seventh aspect: A kit for detecting Corynebacterium glutamicum or its closely related species, comprising the oligonucleotide combination described above (the forward primer shown in EQ ID NO. 33, the reverse primer shown in SEQ ID NO. 5, and the TaqMan fluorescent probe shown in SEQ ID NO. 32), as well as qPCR reaction premix, nuclease-free water, and / or positive control DNA of Corynebacterium glutamicum or its closely related species.

[0028] The qPCR reaction premix was AceQ Universal U+ Probe Master Mix V2.

[0029] Eighth aspect: The use of the oligonucleotide combination or the kit in the preparation of products for diagnosing infections of Corynebacterium glutamicum or its closely related species also falls within the scope of protection of this invention.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This study, through microbiome analysis, found that *Corynebacterium glutamicum* or its closely related species were specifically enriched in the urine of patients with calcium oxalate stones, with a detection rate of approximately 25.8%. Koch's postulate confirmed the pathogenicity of this bacterium for calcium oxalate stones. Mechanistically, we identified hippuric acid as a key stone-inhibiting factor in urine, demonstrating that *Corynebacterium glutamicum* or its closely related species degrade hippuric acid in urine through their encoded hippuric acid hydrolase, disrupting urinary crystallization balance and promoting the precipitation of calcium oxalate crystals. Simultaneously, *Corynebacterium glutamicum* or its closely related species form biofilms that accelerate crystal aggregation. Regarding prevention and treatment strategies, this bacterium exhibits multidrug resistance, limiting the availability of antibiotics. However, hippuric acid supplementation or intervention with polyphenol-rich foods can effectively reverse its pathogenic phenotype. This study is the first to systematically reveal a new mechanism by which Corynebacterium urinaria or its closely related species directly promotes calcium oxalate stones through metabolic regulation and biofilm formation. This not only provides a new perspective for understanding the causes of stones, but also provides new targets (hippurate hydrolase) and potential nutritional intervention strategies for clinical prevention and treatment. Attached Figure Description

[0031] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 To characterize the pathogenic mechanism of calcium oxalate stones induced by Corynebacterium glutamicum or its closely related species; Figure 2 Species composition analysis of urine microorganisms in patients with kidney stones and healthy individuals; Figure 3 To identify the pathogenic bacteria causing calcium oxalate stones; a. Distribution and relative abundance of characteristic microorganism A in different populations; b. Lefse analysis of urinary microorganisms in stone patients and healthy individuals; c. Characteristic microorganisms Corynebacterium glucuronolyticum ; Figure 4 To verify the specificity of qPCR technology, amplification curves were generated. Figure 5Treatment regimens for calcium oxalate stones induced by characteristic microorganisms; a. Flowchart of animal experiments on the treatment of Corynebacterium glutamicum infection with hippuric acid and cranberry diet, with the experiment set up a control group (PBS, n=8), a C. g group (C. g, n=8) and a hippuric acid / cranberry treatment group (C. g+HA / CB, n=8); b. Microscopic examination of calcium oxalate crystals in rat urine. Representative urinary calcium oxalate crystal photographs of rats in each group on day 21. Agg. ratio refers to the proportion of rats in the group with obvious calcium oxalate crystal aggregation in their urine. c. qPCR detection of microbial colonization in rat urine; d. 24-hour urinary hippuric acid content of rats in each group on day 21; e. 24-hour serum hippuric acid content of rats in each group on day 21. Data are presented as Mean ± SEM (PBS, n=7; C. g, n=7; C. g+Rif, n=8). Statistical differences were calculated using Tukey's multiple comparisons test (p<0.0001). p<0.001, (p>0.05, ns); Figure 6 To verify the pathogenicity of *Corynebacterium glutamicum* or its closely related species using Koch's postulates: a. Schematic diagram of animal model verification; b. qPCR results of rat bladder urine against *Corynebacterium glutamicum* or its closely related species; c. Effect of injection of *Corynebacterium glutamicum* or its closely related species on the formation and aggregation of calcium oxalate crystals in rat urine (representative images are shown here; Agg. Ratio indicates the proportion of rats with obvious calcium oxalate crystal aggregates in urine within the same group); d. Scanning electron microscopy observation of urinary liquid crystal aggregates; e. Localization of GFP-labeled *Corynebacterium glutamicum* N11 in rat urinary liquid crystal aggregates after infection. Figure 7 To investigate how *Corynebacterium glutamicum* promotes the development of calcium oxalate stones by degrading hippuric acid, the following study was conducted: a. Urine and serum hippuric acid levels were measured in rats in the PBS, Cg-1, and Cg-2 groups. Data are presented as mean ± SEM (n=8), and statistical differences were calculated using Tukey's multiple comparisons test (p<0.01). b. Measurement of urinary and serum hippuric acid levels in healthy subjects and patients with calcium oxalate stones. Data are presented as mean ± SEM (n=30), and statistical differences were calculated using the Mann-Whitney test (p<0.001, ns). (p>0.05, ns); c. The ability of different strains of Corynebacterium glutamicum to degrade hippuric acid in human urine; Figure 8 The effect of HA on CaOx crystallization. a. Optical microscope image (scale bar: 50 μm); b. and scanning electron microscope image (scale bar: 10 μm); c. Changes in the aspect ratio of CaOx

[100] /

[010] (i.e., a / b); blank group n=309, HA group n=201. Each bar represents the mean of three independent batch experiments. Data are expressed as Mean ± SEM; statistical significance was determined by Student's t test (p<0.01); d. Comparison of CaOx crystal number density. Three replicates were set for each group, and six fields of view were randomly selected for each replicate to calculate the average number of crystals per unit area. Data are expressed as Mean ± SEM. Statistical significance was determined by Student's t test (p<0.01); e. Atomic force microscope image of the (001) plane of CaOx crystal (scale bar: 200 nm); Figure 9 To investigate the effects of hippuric acid and *Corynebacterium glucolyticum* in human urine on calcium oxalate crystal formation in an in vitro experiment, 10 fields of view were randomly selected for each reaction system, and the number of calcium oxalate crystals in each field of view was statistically analyzed. Data are presented as mean ± SEM (n=10), and statistical differences were calculated using the Kruskal-Wallis test (p<0.0001). p<0.01, p<0.1, (p>0.05, ns); Figure 10 Maps of the pK18mobsacB plasmid backbone and the pK18-Ptuf-Gm-mobsacB plasmid backbone. Figure 11 The plasmids used to construct hippuric acid hydrolase knockout strains M2 and M3, and the PCR validation results; Figure 12 To investigate the hippuric acid degradation capacity of Corynebacterium glucoside N11 and its hippuric acid hydrolase knockout strains M2 and M3, three biological replicates were set up for each group, and the data are expressed as mean ± SEM. Figure 13Functional validation of hippuric acid hydrolase knockout strains in animal models: a. Animal experimental procedure for urinary tract injection of *Corynebacterium glucuronolyticum* strain N11 and its hippuric acid hydrolase knockout strain M3; b. Characteristics of calcium oxalate crystals in the urine of experimental rats, representative images of crystal aggregation characteristics in the urine of *C. glucuronolyticum* N11, M3 and control rats observed under a microscope, Agg. Ratio indicates the proportion of rats with obvious calcium oxalate crystal aggregates in the urine within the same group; c. Hippuric acid content in rat urine (left) and serum (right); Figure 14 To investigate the role of *Corynebacterium glutamicum* or its closely related species in promoting calcium oxalate crystal aggregation via biofilm: a. Microscopic observation of the effects of different strains on calcium oxalate crystal aggregation; b. Effects of live, dead, and culture supernatants of *Corynebacterium glutamicum* strain N11 on calcium oxalate crystal aggregation; c. Scanning electron microscopy characterization of calcium oxalate crystals and live-bacterium glutamicum strain N11-calcium oxalate crystal aggregates; d. Crystal violet assay to test the biofilm-forming ability of different strains in human urine; e. Correlation analysis of calcium oxalate crystal aggregation ability and biofilm-forming ability of clinical stone-derived strains N8, N9, N10, N11, and N12; f. Effect of DNase I on biofilm disruption on the crystal aggregation effect of *Corynebacterium glutamicum* strain N11 (C. g. incubation in the experimental system for 10 days). 6 CFU / mL of Corynebacterium glutamicum N11 was used for Pre-DNase I in BHIY medium supplemented with DNase I, and Post-DNase I was added after Corynebacterium glutamicum N11 was co-incubated with calcium oxalate crystals for 4 h and aggregates were formed.

[0032] Figure 15 The effects of different clinical isolates of Corynebacterium glutamicum or its closely related species on the formation and aggregation of calcium oxalate crystals in rat urine (representative images of urine from each rat in the group are shown here). Detailed Implementation

[0033] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0034] Instructions for strain preservation: Category Naming: Corynebacterium glucuronolyticum; Strain name: Corynebacterium glucuronolyticum N11; Accession number: CCTCC NO: M 2026881; Preservation institution: China Center for Type Culture Collection; Location of collection: Wuhan University, Wuhan, China; Preservation date: April 30, 2026.

[0035] Example 1: Identification of pathogenic microorganisms for calcium oxalate stones This embodiment illustrates the screening and identification of pathogens closely related to the formation of calcium oxalate kidney stones, providing key pathogen targets for elucidating the pathogenic mechanism and developing targeted therapy strategies.

[0036] 1. Experimental Design and Methods To identify the microorganisms associated with calcium oxalate stones, we collected morning midstream urine samples from 32 patients with calcium oxalate stones and 28 healthy volunteers. The microbial composition of the urine samples was analyzed using 5R 16S rRNA sequencing. In terms of species composition, *Corynebacterium* was more abundant in the urine of stone patients. Figure 2 LEfSe analysis revealed that *Corynebacterium glycoside*, a characteristic bacterium, was enriched in patients with gallstones. Figure 3 b), and the relative abundance and distribution of this bacterium in patients ( Figure 3 a) Both were significantly higher than the healthy control group, suggesting that Corynebacterium glutamicum may be associated with the occurrence and development of calcium oxalate stones.

[0037] 2. Isolation and universality verification of the target microorganism We further followed Koch's postulates to verify the influence of *Corynebacterium glutamicum* on the occurrence and development of calcium oxalate stones. We first successfully isolated *Corynebacterium glutamicum* strains from urine samples of stone patients in 45.5% (5 / 11) of whom had *Corynebacterium glutamicum* detected by 5R sequencing. Figure 3 c), named N8, N9, N10, N11, and N28, respectively. The 16S rRNA gene sequence alignment results of these isolated strains are as follows: The 16S rRNA gene sequence of strain N8 is shown in SEQ ID NO.1; The 16S rRNA gene sequence of strain N9 is shown in SEQ ID NO.2; The 16S rRNA gene sequence of strain N10 is shown in SEQ ID NO.3; The 16S rRNA gene sequence of strain N11 is shown in SEQ ID NO.4; The 16S rRNA gene sequence of strain N28 is shown in SEQ ID NO.3.

[0038] The sequences were BLAST-aligned with those in the GenBank database. The results showed that the 16S rRNA gene sequences of strains N8, N10, and N28 were consistent with those of strains N10 and N28. Corynebacterium glucuronolyticum The whole genome sequence of DSM 44120 (GenBank accession number: CP047452.1) showed 100% similarity, and the 16S rRNA gene sequence of strain N11 was similar to... Corynebacterium glucuronolyticum The whole genome sequence of DSM 44120 (GenBank accession number: CP047452.1) showed 99.67% similarity, and the 16S rRNA gene sequence of strain N9 was similar to... Corynebacterium glucuronolyticum The whole genome sequence of DSM 44120 (GenBank accession number: CP047452.1) has a similarity of 99.28%.

[0039] Further whole-genome sequencing was performed using the Illumina and Oxford-Nanopore hybrid sequencing platform, obtaining approximately 2.9 Mb of complete genomes for each strain. The sequence numbers for strains N8, N9, N10, N11, and N28 are LMSG_G000064419.1, LMSG_G000064420.1, LMSG_G000064421.1, LMSG_G000064422.1, and LMSG_G000064423.1, respectively (eLibrary of Microbial Systematics and Genomics). Strains N8, N10, N11, and N28 are related to... C. glucuronolyticum The average nucleotide identity (ANI) of DSM 44120 was higher than 97.7%, classifying it as *Corynebacterium glucoside*. Corynebacterium glucuronolyticum The ANI value of strain N9 was 87.22%, lower than the species classification threshold (95%–96%), suggesting that it belongs to a newly emerging closely related species of *Corynebacterium glutamicum*. Given that N9 and... C. glucuronolyticum Based on the greater than 99% 16S rRNA similarity of DSM 44120 and the experience of species nomenclature in related studies, N9 is reclassified as a new species and named Corynebacterium paraglucoside. (Corynebacterium) paraglucuronolyticum) In this invention, *Corynebacterium glucoside* (…) C. glucuronolyticum ) and its closely related species (such as Corynebacterium paraglucosidase) C. paraglucuronolyticum They are collectively referred to as "Corynebacterium glucoside or its closely related species".

[0040] Given that strain N11 has a highly consistent genetic background with the model strain DSM 44120 and is easier to genetically modify, it was preserved as a representative strain for subsequent research on pathogenic mechanisms and control strategies. Corynebacterium Glucoside N11 is denoted as Corynebacterium glucuronolyticum N11, this strain was deposited at the China Center for Type Culture Collection on April 30, 2026, with accession number: CCTCC NO:M 2026881.

[0041] To determine the generalizability of *Corynebacterium glucolyticus* or its closely related species in the urine of patients with kidney stones, we developed a TaqMan quantitative real-time PCR method for detecting *Corynebacterium glucolyticus* or its closely related species in urine samples, enabling rapid, sensitive, and specific detection. Using this method, we performed qPCR detection of *Corynebacterium glucolyticus* or its closely related species in midstream urine samples from 62 patients with calcium oxalate stones and 67 healthy individuals. The results showed that *Corynebacterium glucolyticus* or its closely related species were detectable in the urine of 25.8% of the stone patients, while they were not present in the healthy control group. This confirms that *Corynebacterium glucolyticus* or its closely related species can indeed be found in the urine of stone patients. This result demonstrates a strong association between *Corynebacterium glucolyticus* or its closely related species and the disease state of calcium oxalate stones.

[0042] The development of qPCR detection technology for Corynebacterium glucoside or its close relatives includes the following steps: 1) Collection of urine samples and DNA extraction 50 mL of the subject's first morning urine was collected and centrifuged at 12,000 rpm for 20 min at 4°C within 1 h. The supernatant was discarded, and the precipitate was collected. DNA was extracted using a urine DNA extraction kit (Absin, Shanghai, China, Cat# abs60291-100T), and finally washed with 30-50 μL of nuclease-free water preheated to 56°C. The concentration of the extracted DNA was determined using a Qubit 2.0 fluorometer (Life Technologies, USA), and the DNA was either directly used for subsequent qPCR or frozen at -80°C for later use.

[0043] 2) Establishment of the TaqMan qPCR detection system (1) Primers and probes The partial specific sequence of the marker gene dTDP-4-dehydrorhamnose 3,5-epimerase, which has actual physiological function in Corynebacterium daunoside or its closely related species, was selected as the target amplification sequence, and primers and fluorescent probes were designed.

[0044] The target sequence of the dTDP-4-dehydrorhamnose 3,5-epimerase gene is shown in SEQ ID NO.31; TaqMan Probe(5'-FAM, 3'-MGB):CACAGCCTCTACCTTCCCGTC; (SEQ ID NO.32) Forward primer (SEQ ID NO.33): GTAGGCGACGGTGGAATCTT; Reverse primer (SEQ ID NO.5): GTTTCGGACGCTACATCGGT; (2) qPCR reaction system qPCR was performed using AceQ Universal U+ Probe Master Mix V2 (Vazyme, Nanjing, China), and the 20 μL system is shown in Table 1: Table 1

[0045] (3) qPCR amplification program qPCR was performed using the QuantStudio™ 1 Real-Time PCR Instrument, and the amplification program is shown in Table 2: Table 2

[0046] 3) Detection specificity verification respectively C. glucuronolyticum (Cg) and common urinary tract microorganisms Escherichia coli (Ec) Pseudomonas putida (Pp) Lactobacillus acidophilus (La) bacterial DNA was used as a template for qPCR specificity verification. Figure 4 As shown, only C. glucuronolyticum The presence of a specific amplification curve indicates that the invention has good specificity, while other strains showed no obvious amplification signal.

[0047] 4) Clinical sample testing The method of this invention was used to detect calcium oxalate stones in urine samples from patients and controls. A positive result was defined as Ct < 37 when the urine DNA concentration was less than 100 ng / μL and Ct < 35 when the urine DNA concentration was greater than or equal to 100 ng / μL. The results are shown in Table 3: 25.81% (16 / 62) of the samples in the stone group were detectable. C. glucuronolyticum It and its closely related species, while no detection was found in the control group samples (0 / 67). C. glucuronolyticum and its closely related species.

[0048] Table 3

[0049] Example 2: Exploration of the pathogenicity of *Corynebacterium Gluconopsis* or its close relatives to calcium oxalate stones. To assess the pathogenicity of Corynebacterium glutamicum or its close relatives to calcium oxalate stones, we used the formation and aggregation of calcium oxalate crystals in urine as an indicator of the occurrence and development of calcium oxalate stones, and constructed an infection model by urinary tract injection.

[0050] 1. Laboratory animals and grouping: To systematically evaluate the pathogenicity of *Corynebacterium glutamicum* or its closely related species and the prevalence among strains, we conducted two independent phases. Adult male SD rats were used in both phases, and were pretreated with 1.5% ethylene glycol in their drinking water 7 days prior to infection. Rats were randomly divided into groups of eight. All groups infected with *Corynebacterium glutamicum* or its closely related species underwent bladder instillation of a bacterial suspension of the corresponding strain (10... 9 CFU / animal, 0.1 mL / time, once daily for 7 days), with an equal volume of sterile phosphate-buffered saline (PBS) instilled into the bladder once daily for 7 days as the negative control group. Figure 6 a).

[0051] The first phase aimed to preliminarily assess the pathogenicity of *Corynebacterium glutamicum* or its close relatives, and two infection groups were established: Strain N11 (C. g-1 group): Strain N11 isolated in Example 1; The model strain of *Corynebacterium Glucoside* (C. g-2 group): DSM 44120; The second phase aimed to assess whether Corynebacterium Glucoside Alternaria or its closely related species are generally pathogenic, and four infection groups were set up: Strain N8 (N8 group): Strain N8 isolated in Example 1; Strain N9 (N9 group): Strain N9 isolated in Example 1; Strains N10 (N10 group): Strains N10 isolated in Example 1; Strain N28 (N28 group): Strain N28 isolated in Example 1; 2. *Corynebacterium glutamicum* or its close relatives promote the formation and aggregation of calcium oxalate crystals. qPCR detection of microorganisms in rat urine on day 15 showed that various *Corynebacterium glucoside* species or their closely related species could successfully colonize the rat urinary tract. Compared with the PBS group ( Figure 6 b) In the urine of rats infected with both the model strain and clinical strain N11 of Corynebacterium glutamicum, the number of calcium oxalate crystals was significantly increased and obvious aggregation occurred. Figure 6c), indicating that *Corynebacterium glutamicum* promotes the occurrence and development of calcium oxalate stones by promoting the formation and aggregation of calcium oxalate crystals. Scanning electron microscopy observation of these crystal aggregates in rat urine revealed a large number of rod-shaped microorganisms adhering between the crystals. Figure 6 d), we then infected rats with *Corynebacterium glutamicum* N11 labeled with superfold GFP (sfGFP), and found that green fluorescence could be observed in calcium oxalate crystal aggregates in the rat urine. Figure 6 e), thus demonstrating that *Corynebacterium glucoside* directly participates in the aggregation of calcium oxalate crystals in rat urine, playing a crucial role in stone formation. Furthermore, animal experiments confirmed that all clinically derived *Corynebacterium glucoside* strains or their closely related strains (including N8, N9, N10, and N28) can promote the formation and aggregation of calcium oxalate crystals in rat urine. Figure 15 This confirms that the pathogenicity of Corynebacterium glucoside or its closely related species is universal among different strains of this bacterium.

[0052] In summary, *Corynebacterium glutamicum* or its close relatives have the ability to promote the formation and aggregation of calcium oxalate crystals, and are a pathogenic bacterium that induces calcium oxalate stones.

[0053] Example 3: Exploring the mechanism by which Corynebacterium Glucono-deltae or its closely related species promotes stone formation through hippuric acid degradation. 1. The mechanism by which *Corynebacterium glutamicum* or its closely related species promotes stone formation is closely related to urinary hippuric acid levels. To explore the pathogenic mechanism of *Corynebacterium glutamicum* or its close relatives in inducing calcium oxalate stones, urine samples were collected from rats in the PBS and C. g-1 groups. Non-target metabolomics analysis was performed using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). The results showed that the C. g-1 group shared nine core differential metabolites with the PBS group: N-arachidonicylglycine, Xi-3-hydroxy-5-phenylpentanoic acid O-β-D-glucopyranoside, (S)-α-amino-4-carboxy-3-furanopropionic acid, flavoxol glucoside, limonene, isoflavonic acid 3-O-glucuronide, hippuric acid, N5-(3,4-dioxo-1,5-cyclohexadien-1-yl)-L-glutamine, and norsine.

[0054] By cross-referencing the aforementioned differentially metabolites with data from several published studies of urinary metabolomics in patients with kidney stones versus healthy individuals (X. Wang) et al., Identification of urine biomarkers for calcium-oxalate urolithiasis in adults based on UPLC-Q-TOF / MS. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 1124, 290-297 (2019); X. Duan et al. , 1H NMR-based metabolomic study of metabolic profiling for the urine of kidney stonepatients. Urolithiasis 48, 27-35 (2020); C. Thongprayoon et al. , Nuclearmagnetic resonance metabolomic profiling and urine chemistries in incidentkidney stone formers compared with controls. J. Am. Soc. Nephrol. In our study (33, 2071-2086 (2022)), we found that hippuric acid was the only common differentially expressed metabolite. Subsequently, LC-MS analysis revealed that urinary hippuric acid levels in rats from *Corynebacterium glucolyticum* or its closely related species (C. g-1 and C. g-2 groups) were significantly lower than in the control group, but blood hippuric acid levels showed no significant difference compared to the control group. This indicates that the decrease in urinary hippuric acid levels was not caused by systemic metabolic changes. Therefore, we hypothesize that this change is more likely due to alterations in the urinary microenvironment caused by colonization of *Corynebacterium glucolyticum* or its closely related species. Figure 7 a). We then tested the blood and urine hippuric acid levels in 30 patients with calcium oxalate stones and healthy individuals, and found that the clinical data showed the same pattern as the blood and urine hippuric acid levels in rats with *Corynebacterium glutamicum* or its closely related species (a). Figure 7 (b) That is, the level of hippuric acid in the urine of patients with calcium oxalate stones was significantly lower than that in the healthy control group, while there was no significant difference in the blood hippuric acid level between stone patients and healthy people.

[0055] This study found that different strains of Corynebacterium glucoside or its closely related species all exhibited strong hippuric acid hydrolysis ability in human urine. Figure 7 c) indicates that the hippuric acid degradation ability of Corynebacterium glucoside or its close relatives is universal among different strains of this bacterium. Therefore, it is speculated that Corynebacterium glucoside or its close relatives may affect the occurrence and development of calcium oxalate stones by reducing the hippuric acid content in urine.

[0056] 2. In vitro verification of hippuric acid inhibiting calcium oxalate crystal formation We first simulated the formation of calcium oxalate crystals in vitro by sequentially adding CaCl2 and Na2C2O4 solutions to a NaCl solution, ultimately forming a reaction system of 0.7 mM CaCl2, 0.7 mM Na2C2O4, and 150 mM NaCl (total volume 20 mL). A clean glass slide was placed at the bottom of the reaction vessel to collect the crystals for subsequent observation. To assess the effect of hippuric acid on crystallization, hippuric acid was added to a final concentration of 2 mM (the concentration of hippuric acid in healthy human urine) before adding sodium oxalate. After standing at 60°C for 24 h, the slide was removed, dried at 25°C, and the basic characteristics such as the number, size, and surface morphology of the calcium oxalate crystals were characterized.

[0057] Calcium oxalate crystals typically have a hexagonal prismatic shape. (Optical microscope) Figure 8 a) and scanning electron microscope ( Figure 8 b) Observation of the number of calcium oxalate crystals showed that the crystal density per unit area of ​​the hippuric acid group ( Figure 8 a, Figure 8 d) and the aspect ratio

[100] /

[010] of the CaOx crystal are both significantly reduced ( Figure 8 b、 Figure 8 c) indicates that hippuric acid significantly inhibits the nucleation and longitudinal growth of calcium oxalate crystals. Subsequently, atomic force microscopy (AFM) topological imaging of the crystal surface revealed that the hippuric acid group exhibited higher surface roughness and deeper dissolution pits. Figure 8 e). In summary, the results confirm that hippuric acid is indeed an inhibitor of calcium oxalate stones.

[0058] 3. Verification of the effect of *Corynebacterium Glucono-delta-lactamase* or its close relatives on promoting crystal formation by degrading hippuric acid. To better simulate the physiological environment, we collected urine samples from six patients with kidney stones. After centrifugation at 8000 rpm for 10 min, we collected the supernatant and induced calcium oxalate crystal formation in vitro at 37°C. Each urine sample was divided into four groups: (1) Untreated urine supernatant control (N); (2) Urine supernatant with 2 mM hippuric acid (N+HA); (3) Incubate Corynebacterium glucoside (strain N11) in urine supernatant containing 2 mM hippuric acid for 48 hours, and centrifuge to obtain sterile supernatant (N+HA+C. g-1).

[0059] (4) Incubate Corynebacterium Glucoside (strain DSM 44120) in urine containing 2 mM hippuric acid for 48 hours, and centrifuge to obtain sterile supernatant (N+HA+C. g-2).

[0060] All samples were adjusted to pH 5.8 and filtered through a 0.22 μm filter membrane before use. Sterile Na₂C₂O₄ and CaCl₂ were added to each sample to final concentrations of 0.5 mM and 5 mM, respectively, followed by incubation at 37°C for 2–3 hours. CaOx crystals were imaged using an inverted microscope, with 10 fields of view randomly selected for each group. The number of crystals in each field of view was quantified using ImageJ software.

[0061] The results showed that the addition of 2 mM hippuric acid significantly inhibited the formation of calcium oxalate crystals in urine. Figure 9 Urine supplemented with 2 mM hippuric acid was co-cultured with *Corynebacterium glutamicum*, and the supernatant was used for calcium oxalate crystal formation experiments. The results showed that compared to the control group without *Corynebacterium glutamicum* culture, the number of crystals generally increased, with 80% (4 / 5) of the patients' urine samples returning to the crystal formation level without hippuric acid supplementation. Figure 9 These results indicate that hippuric acid inhibits CaOx crystal formation, while *Corynebacterium glucoside* promotes crystal formation by degrading hippuric acid in urine.

[0062] 4. Construction and animal model validation of hippuric acid hydrolase gene knockout strain M3 Given that Example 2 has confirmed that different types of Corynebacterium glutamicum or their closely related species all possess hippuric acid hydrolysis capabilities, and that this capability is closely related to the occurrence of stones, this example uses the representative strain N11 as a background to construct a hippuric acid hydrolase gene knockout mutant to verify the key role of this enzyme in the pathogenesis process.

[0063] Using the *Corynebacterium glutamicum* N11 isolated in Example 1 as the starting strain, a mutant strain with double gene knockout (hippuric acid hydrolase genes 1255 and 8260) and carrying sfGFP fluorescent label was constructed and named M3.

[0064] Vector backbone: suicide plasmid pK18mobsacB (image shown) Figure 10 After modification, that is, while retaining its original resistance marker, the gentamicin resistance gene GmR (SEQ ID NO.7), driven by the endogenous promoter Ptuf (SEQ ID NO.6) of Corynebacterium glutamicum, was introduced to form the basic vector pK18-Ptuf-Gm-mobsacB suitable for this study, as shown in the figure. Figure 10 As shown in the figure.

[0065] The specific steps are as follows: 1) Construction of knockout vectors pK18-Δ1255 and pK18-Δ8260::sfGFP Using the genomic DNA of strain N11 as a template, and with the primers listed in Table 4, the upstream and downstream homologous arm fragments (approximately 1.5 kb each) of the hippuric acid hydrolase genes 1255 and 8260 were amplified by PCR.

[0066] The upstream homologous arm fragment of hippuric acid hydrolase gene 1255 is shown in SEQ ID NO.8; the downstream homologous arm fragment of hippuric acid hydrolase gene 1255 is shown in SEQ ID NO.9.

[0067] The upstream homologous arm fragment of hippuric acid hydrolase gene 8260 is shown in SEQ ID NO.10; the downstream homologous arm fragment of hippuric acid hydrolase gene 1255 is shown in SEQ ID NO.11.

[0068] Simultaneously, the sfGFP gene (SEQ ID NO.28) was placed under the control of another endogenous strong promoter, PdnaK (SEQ ID NO.12), to construct the PdnaK-sfGFP expression cassette. All PCR products were purified using a gel extraction kit (Omega Bio-tek, Norcross, USA), and then, using Gibson Assembly® Master Mix (New England Biolabs, Ipswich, USA), homologous arms of the 1255 gene or the 8260 gene were assembled with the PdnaK-sfGFP expression cassette into the pK18mobsacB vector, which had been linearized by double digestion with XhoI and XmaI, to construct the homologous recombination knockout vectors pK18-Δ1255 and pK18-Δ8260::sfGFP.

[0069] 2) Two-step method for constructing dual-gene knockout strain M3 We found that for C. glucuronolyticum N11 strain, Cg_1255 (SEQ ID NO.29) is C. glucuronolyticum The primary hippuric acid hydrolase encoding gene, Cg_8260 (SEQ ID NO.30), is a secondary hippuric acid hydrolase encoding gene.

[0070] Step 1: Constructing the single-gene knockout strain M2 (Δ1255) The knockout vector pK18-Δ1255 was electroporated into *Corynebacterium glutamicum* strain N11. Transformants were plated on BHIY plates containing gentamicin (20 mg / L) and incubated upside down at 37°C for 2–3 days. Clones that underwent the first homologous recombination (single crossover), integrated the plasmid into the chromosome, and acquired resistance were screened.

[0071] To identify recombinants undergoing double crossover, single clones were picked and further cultured on plates containing 10% sucrose. Reverse selection was performed using the sacB (SEQ ID NO. 13) gene from the vector to promote a second homologous recombination (double crossover). Subsequently, single clones were picked using sterile toothpicks and streaked onto BHIY agar plates containing gentamicin and those without antibiotics. After incubation at 37°C for 1-2 days, colonies that grew on antibiotic-free plates but not on gentamicin-containing plates were selected (indicating loss of the plasmid backbone). Finally, colony PCR was used to verify the deletion of the 1120 gene, obtaining the correct mutant strain, named M2. Figure 11 The plasmids used to construct this strain and the validation process.

[0072] Step 2: Construct a double knockout strain M3 (Δ1255, Δ8260::sfGFP) based on M2. The knockout vector pK18-Δ8260::sfGFP was electroporated into the intermediate strain M2. The same selection strategy as in the first step (primary screening for gentamicin resistance, followed by sucrose reverse selection and gentamicin sensitivity verification) was used to obtain recombinants with double crossover. Colony PCR confirmed that the 8260 gene was replaced by the PdnaK-sfGFP expression cassette, and that the Δ1255 background of M2 was preserved. The final strain with double gene knockout and carrying the sfGFP fluorescent marker was named M3. Figure 11 (The plasmids used to construct this strain and the validation process).

[0073] Table 4 Primer information used for strain construction

[0074] We then tested the single knockout strain (M2) and the chassis strain (Cg_1255) of Cg_1255 in BHIY medium supplemented with 20 mM hippuric acid. C. glucuronolyticum The hippuric acid hydrolysis capacity of N11 and Cg_8260 double-knockout strains (M3) was compared with that of the chassis strain. C. glucuronolyticum Compared to N11, M2 hardly degraded hippuric acid at 24 h, but with prolonged time, this strain was able to completely degrade 20 mM hippuric acid at 48 h. M3, however, completely lost its ability to degrade hippuric acid. Figure 12 This indicates that strain M3, with complete knockout of hippuric acid hydrolase, was successfully constructed.

[0075] 3) Animal phenotype Following the method described in Example 2, the following rat experiments were conducted. ① PBS control group: The bladder was instilled with an equal volume of sterile phosphate-buffered saline; ② Corynebacterium Glucoside N11 infection group (N11 group): Bladder instillation of Corynebacterium Glucoside N11 wild-type bacterial suspension (10 9 CFU / animal, 0.1 mL / dose); ③ Mutant strain M3 infection group (M3 group): Bladder instillation of knockout strain M3 bacterial suspension (10 9 CFU / animal, 0.1 mL / dose).

[0076] For 7 consecutive days, rats were injected urethra with the corresponding bacterial suspension or PBS. After infection, the urinary liquid crystal phenotype of each group of rats was observed, and the levels of hippuric acid in urine and blood were measured. Figure 13 a). The results showed that, compared with the starting strain Corynebacterium glutamicum N11, the M3 strain had a lower probability of observing calcium oxalate crystal aggregation in the urine of rats treated (calcium oxalate crystals were observed in only 50% of rat urine), and the aggregation area of ​​the crystals was smaller. Figure 13 b), and the urinary hippuric acid level in the M3 group rats was significantly higher than that in the original bacteria N11 group, even approaching the normal level (i.e., the PBS group). Figure 13 c) indicates that hippuric acid hydrolase is a key pathogenic factor in the induction of calcium oxalate stones by *Corynebacterium glucoside* or its close relatives. In summary, *Corynebacterium glucoside* or its close relatives degrade hippuric acid in urine through their produced hippuric acid hydrolase, leading to increased precipitation of calcium oxalate crystals and thus promoting the occurrence and development of calcium oxalate stones.

[0077] Example 4: Exploring the mechanism by which Corynebacterium Glucoside-Degrading and its closely related bacteria promote crystal aggregation through biofilm. Given the observation of a large amount of calcium oxalate crystals aggregated in rat urine, we also explored the aggregation mechanism of calcium oxalate crystals.

[0078] First, prepare a culture medium containing 10... 6 CFU / mL of *Corynebacterium glucolyticus* N11 (C. g-1) and the model strain DSM44120 (C. g-2) was added to 2 mL of N11 and DSM44120 bacterial suspensions and the control group (sterile medium), and incubated at 37°C for 3–6 h. Optical microscopy showed that *Corynebacterium glucolyticus* significantly aggregated calcium oxalate crystals, while the crystals in the blank control group were dispersed. Figure 14 a).

[0079] We then tested the calcium oxalate crystal aggregation ability of live bacteria, dead bacteria, and culture supernatant at equal concentrations using strain N11. The results showed that only the live bacteria group could aggregate crystals. Figure 14(b) ruled out the influence of bacterial extracellular metabolites on crystal aggregation, indicating that the aggregation of calcium oxalate crystals by *Corynebacterium glutamicum* depends on the growth characteristics of the live bacteria, which may be related to the specific attachment structures between live bacteria observed by scanning electron microscopy. Figure 14 c).

[0080] Our experiments have shown that different strains of *Corynebacterium glucoside* or its closely related species all have a strong biofilm-forming ability in human urine. Figure 14 d), and tested the in vitro calcium oxalate crystal aggregation ability of strains N8, N9, N10, N11, and N12 from clinical stone patients, finding a positive correlation between crystal aggregation ability and biofilm formation ability ( Figure 14 e), indicating that the ability of biofilms to form is directly related to the aggregation of calcium oxalate crystals.

[0081] Extracellular DNA is an important component of bacterial biofilm matrix, and DNase I can effectively disrupt bacterial biofilms. We found that not only did DNase I-treated *Corynebacterium glutamicum* N11 (pre-DNase I+) fail to significantly aggregate calcium oxalate crystals, but when DNase I (post-DNase I+) was added to a system that had already formed bacterial-calcium oxalate crystal aggregates, almost all the aggregates were dispersed into free crystals. Figure 14 f). This indicates that disrupting the biofilm can cause *Corynebacterium glutamicum* or its close relatives to lose their ability to aggregate crystals, fully demonstrating the key role of *Corynebacterium glutamicum* or its close relatives in promoting calcium oxalate crystal aggregation through the biofilm.

[0082] In summary, *Corynebacterium glutamicum* or its close relatives degrade hippuric acid in urine through their encoded hippuric acid hydrolase, leading to increased precipitation of calcium oxalate crystals in the urine. Furthermore, these calcium oxalate crystals aggregate through biofilm formation, and the synergistic effect of these two processes promotes the formation and development of calcium oxalate stones. Figure 1 ).

[0083] Example 5: Treatment with hippuric acid supplementation We further sought treatment strategies that could directly increase urinary hippuric acid levels. Hippuric acid is a shared metabolite of the host and gut microbiota. Urinary hippuric acid mainly originates from the binding of benzoic acid and glycine in the liver and the breakdown and metabolism of amino acids such as phenylalanine by gut microbes. Benzoic acid in the liver is largely produced by the degradation of polyphenols in food by gut microbes. Multiple studies have shown that 24-hour urinary hippuric acid can serve as a biomarker for high-polyphenol diets (such as fruits and vegetables). Cranberries are rich in benzoic acid and polyphenols, and long-term consumption of cranberries can safely and effectively increase the levels of hippuric acid in blood and urine. Therefore, we selected two treatment options: direct oral administration of hippuric acid (HA) and a cranberry (CB) diet, to investigate whether hippuric acid supplementation could improve the formation and aggregation of calcium oxalate crystals caused by infection with *Corynebacterium glutamicum* or its closely related species. Figure 5 a).

[0084] 1. Experimental Grouping Following the method in Example 2, a urinary tract infection model of *Corynebacterium glutamicum* or its closely related species was established in SD rats via bladder instillation. The rats were randomly divided into the following groups (n=8 per group): Model control group (C. g group): Bladder instillation of Corynebacterium Glucoside N11 suspension (10 9 CFU / animal, 0.1 mL / time), administered via gavage with regular feed and an equal volume of solvent; Direct hippuric acid supplementation group (HA group): Bladder instillation of Corynebacterium glutamicum N11 suspension (10 9 CFU / animal, 0.1 mL / time), daily gavage administration of sodium hippurate solution (converted according to body weight, the dose is equivalent to the human equivalent therapeutic dose). Cranberry diet intervention group (CB group): Bladder instillation of Corynebacterium glutamicum N11 suspension (10 9 CFU / animal, 0.1 mL / feed), fed with a diet-type feed (mixed at a ratio of 5% w / w) from Wuhan Wanqianjiaxing Biotechnology Co., Ltd. containing standardized freeze-dried cranberry powder (rich in benzoic acid and polyphenols).

[0085] All interventions were initiated on day 1 of infection and continued until the end of the experiment (day 21). On day 21, urine samples were collected from rats in each group, and the viral load of *Corynebacterium glutamicum* was detected by qPCR to confirm whether the colonization status of the pathogen was affected by the supplementation intervention.

[0086] 2. Experimental Results: Corynebacterium Glucoside N11 was able to stably colonize in all intervention groups. Figure 5c), and compared with the C.g group, the characteristics of calcium oxalate stones in the HA and CB groups were significantly improved, that is, the number of calcium oxalate crystals in the urine was less and there was basically no crystal aggregation (c). Figure 5 b). Simultaneously, LC-MS quantitative analysis confirmed that urine samples from the HA and CB groups on day 21 ( Figure 5 d) and the levels of hippuric acid in the blood were significantly higher than those in the control group ( Figure 5 e), which shows that increasing the hippuric acid content in urine can significantly reduce the effect of Corynebacterium glutamicum or its close relatives on the formation and aggregation of calcium oxalate crystals, and suggests that hippuric acid supplementation and consuming cranberries are both treatment options.

[0087] In summary, we found that antibiotic treatment was not ideal for increasing the formation and aggregation of calcium oxalate crystals in urine caused by Corynebacterium glutamicum or its close relatives, while hippuric acid supplementation and cranberry dietary intervention had significant therapeutic effects.

[0088] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that these are merely illustrative examples, and any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of an active ingredient in the preparation of a product for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of *Corynebacterium glutamicum* or its closely related species, characterized in that, The active ingredient is selected from one or more of the following substances: hippuric acid, pharmaceutically acceptable salts of hippuric acid, hippuric acid ester prodrugs, benzoic acid, pharmaceutically acceptable salts of benzoic acid, and substances that can promote the synthesis of hippuric acid in vivo.

2. The use according to claim 1, characterized in that, The substance that can promote the synthesis of hippuric acid in the body is a plant extract rich in hippuric acid, benzoic acid, benzoic acid glycosides or polyphenolic compounds. And / or, pharmaceutically acceptable salts of the benzoic acid include sodium benzoate or potassium benzoate.

3. The use according to claim 2, characterized in that, The plant extract is derived from one or more of the following: cranberry, blueberry, European blueberry, raspberry, cloudberry, redberry, plum, plum, and jujube.

4. A pharmaceutical composition, characterized in that, The composition comprises one or more of the following substances: hippuric acid, a pharmaceutically acceptable salt of hippuric acid, a hippuric ester prodrug, a pharmaceutically acceptable salt of benzoic acid, and a substance capable of promoting the synthesis of hippuric acid in vivo; the composition is used for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of Corynebacterium glutamicum or its close relatives.

5. A kit for the prevention and / or treatment of calcium oxalate kidney stones caused by colonization of *Corynebacterium glutamicum* or its close relatives, characterized in that... The kit contains: a) hippuric acid, its pharmaceutically acceptable salt, or its ester prodrug; or b) Substances that can promote the synthesis of hippuric acid in the body.

6. A method for screening candidate drugs for the prevention and / or treatment of calcium oxalate kidney stones, characterized in that, Includes the following steps: (1) Contact the candidate material with *Corynebacterium glucoside* or its closely related species; and / or its expressed hippuric acid hydrolase; (2) Detect the activity of the hippuric acid hydrolase; If the activity of the hippuric acid hydrolase is inhibited, it indicates that the candidate substance can be used for the prevention and / or treatment of calcium oxalate kidney stones.

7. A qPCR method for detecting *Corynebacterium Glucono-deltae* or its closely related species for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the extracted DNA as a template, qPCR amplification was performed using the forward primer shown in SEQ ID NO.33, the reverse primer shown in SEQ ID NO.5, and the TaqMan fluorescent probe shown in SEQ ID NO.32; (3) Determine whether there is *Corynebacterium glutamicum* or its closely related species in the sample to be tested based on the amplification curve.

8. An oligonucleotide combination for detecting Corynebacterium glucoside or its closely related species, characterized in that, include: The forward primer has the nucleotide sequence shown in SEQ ID NO.33; The reverse primer has the nucleotide sequence shown in SEQ ID NO.5; as well as The TaqMan fluorescent probe has the nucleotide sequence shown in SEQ ID NO.32, and the 5' end of the probe is labeled with a FAM fluorescent reporter group and the 3' end is labeled with an MGB quencher group.

9. A kit for detecting *Corynebacterium glutamicum* or its closely related species, characterized in that, It comprises the oligonucleotide combination as described in claim 8.

10. The use of the oligonucleotide combination of claim 8 or the kit of claim 9 in the preparation of a product for diagnosing infection with Corynebacterium glutamicum or its closely related species.

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